PROJECT SUMMARY The primary goal of this R01 proposal is to characterize the interferon-stimulated gene E74-like ETS transcription factor 1 (ELF1), in vivo and in vitro, regarding its role in restricting virus infections. Although hundreds of interferon-stimulated genes (ISGs) execute the antiviral function of interferon (IFN), for the vast majority, the molecular mechanisms remain a mystery. Understanding the mechanisms by which host factors inhibit viral infections may lead to novel antiviral strategies. The project is founded on our recent published discovery that ELF1 exhibits direct antiviral action in inhibiting influenza A virus. Since that initial discovery, our preliminary data show that ELF1 acts after multiple rounds of viral replication and is broadly antiviral. In epithelial cells, ELF1 transcriptionally regulates a vast program of hundreds of genes mostly distinct from those regulated by IFN. ELF1 does not feed forward to produce IFN, and retains its strong antiviral potential even in the absence of further IFN signaling. Intriguingly, at least part of this program confers protection to neighboring cells. Finally, we show that local knockdown of murine Elf1 during IAV infection significantly increases viral lung titers, morbidity, and mortality in vivo. These results opened the door to propose a novel concept for ELF1's role in the IFN response: that this transcription factor is a novel regulator of inflammation and innate antiviral immunity. In addition, our data raise the exciting possibility that ELF1's antiviral program continues protecting cells from viral infections even in the refractory phase post IFN exposure, or when IFN signaling is abrogated by viral antagonists. The main technical innovation of this proposal is the use the versatile tool of confocal high-content imaging to study sub-cellular signaling events during viral infections. Completion of our aims will have major impact on the field, by characterizing a novel transcriptional antiviral program that is as broad and potent, but distinct from IFN.